Entry of herpes simplex virus (HSV) into cells requires binding of viral glycoprotein gD to one of its receptors which then leads to virus-cell fusion. It is known that HSV penetration is blocked by treating cells bound with virus with a buffer at pH 3. To gain further insights into the molecular mechanism of HSV entry, the effects of acid inactivation were examined using a beta-galactosidase reporter assay. Cell-free virions of HSV-1 (KOS) that were pretreated with mildly acidic pH were incompetent for entry into Vero cells. Thus, low pH directly affects the virions themselves. Specifically, entry activity was inhibited when virions was treated with pH < 6.5. Inactivation was rapid, irreversible and temperature-dependent. Acid-treated virions also failed to enter CHO cells expressing the gD-binding receptors herpesvirus entry mediator A (HveA), nectin-1 or nectin-2. We demonstrated that these receptors mediate stable, heparin-resistant binding of virus to cells at 4?C, and that binding was blocked by addition of soluble gD or soluble receptors. These findings suggest that receptors are essential for entry partly due to their role in strengthening HSV adhesion to cells prior to virus-cell fusion. Low pH treatment did not affect stable binding of HSV to HveA, nectin-1 or nectin-2, nor did it alter the receptor-binding conformation of gD. Taken together, the data indicate low pH perturbs a step in the HSV entry process other than the gD-receptor interaction. We propose that low pH pretreatment incapacitates HSV for fusion with cell membranes and is a useful tool to uncouple the entry processes of receptor binding and fusion.